scholarly journals Functional characterization of the alanine-serine-cysteine exchanger of Carnobacterium sp AT7

2019 ◽  
Vol 151 (4) ◽  
pp. 505-517 ◽  
Author(s):  
Paola Bartoccioni ◽  
Joana Fort ◽  
Antonio Zorzano ◽  
Ekaitz Errasti-Murugarren ◽  
Manuel Palacín
Keyword(s):  
Amino Acid ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Functional Characterization ◽  
Physiological Role ◽  
Cell Uptake ◽  
Amino Acid Transporters ◽  
Inhibitor Selectivity ◽  
Substrate Binding Sites

Many key cell processes require prior cell uptake of amino acids from the environment, which is facilitated by cell membrane amino acid transporters such as those of the L-type amino acid transporter (LAT) subfamily. Alterations in LAT subfamily amino acid transport are associated with several human diseases, including cancer, aminoacidurias, and neurodegenerative conditions. Therefore, from the perspective of human health, there is considerable interest in obtaining structural information about these transporter proteins. We recently solved the crystal structure of the first LAT transporter, the bacterial alanine-serine-cysteine exchanger of Carnobacterium sp AT7 (BasC). Here, we provide a complete functional characterization of detergent-purified, liposome-reconstituted BasC transporter to allow the extension of the structural insights into mechanistic understanding. BasC is a sodium- and proton-independent small neutral amino acid exchanger whose substrate and inhibitor selectivity are almost identical to those previously described for the human LAT subfamily member Asc-1. Additionally, we show that, like its human counterparts, this transporter has apparent affinity asymmetry for the intra- and extracellular substrate binding sites—a key feature in the physiological role played by these proteins. BasC is an excellent paradigm of human LAT transporters and will contribute to our understanding of the molecular mechanisms underlying substrate recognition and translocation at both sides of the plasma membrane.

scholarly journals Molecular and Functional Characterization of a Family of Amino Acid Transporters from Arabidopsis

2004 ◽  
Vol 136 (2) ◽  
pp. 3104-3113 ◽  
Author(s):  
Yan-Hua Su ◽  
Wolf B. Frommer ◽  
Uwe Ludewig
Keyword(s):  
Amino Acid ◽  
Functional Characterization ◽  
Amino Acid Transporters

scholarly journals Yeast Cell-Based Transport Assay for the Functional Characterization of Human 4F2hc-LAT1 and ‐LAT2, and LAT1 and LAT2 Substrates and Inhibitors

2021 ◽  
Vol 8 ◽  
Author(s):  
Satish Kantipudi ◽  
Dimitrios Fotiadis
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Functional Characterization ◽  
Methylotrophic Yeast ◽  
Cell Types ◽  
Amino Acid Transporters ◽  
Cell Assays ◽  
Transport Assay ◽  
Compound Screening

In mammalian cells, the L-type amino acid transporters (LATs) LAT1 (SLC7A5) and LAT2 (SLC7A8) form heterodimeric amino acid transporters (HATs) with the ancillary protein 4F2hc and are involved in the cellular uptake of specific amino acids. The HAT 4F2hc-LAT1 is found upregulated in various cancer cell types, while 4F2hc-LAT2 is a transporter for non-cancer cells. Preclinical studies have highlighted that 4F2hc-LAT1 plays an important role in tumor progression representing a valid anticancer target. Consequently, current research is focusing on the development of potent and specific human 4F2hc-LAT1 inhibitors. On the other hand, 4F2hc-LAT2 is emerging as target of other diseases, thus also gaining clinical interest. To determine affinity and specificity of substrates and inhibitors for 4F2hc-LAT1 or 4F2hc-LAT2, robust transport cell assays are indispensable. We have optimized and validated a transport assay using cells of the methylotrophic yeast Pichia pastoris stably overexpressing the human HATs 4F2hc-LAT1 or -LAT2, and the LATs LAT1 or LAT2 alone. The radioligand [3H]L-leucine was used as reporter and the substrates L-leucine, triiodothyronine (T3) and thyroxine (T4) as well as the inhibitors BCH and JPH203 (KYT-0353) for assay validation. Obtained half-maximal inhibitory concentrations also provided new insights, e.g., into the LAT specificity of the potent inhibitor JPH203 and on the potency of the thyroid hormones T3 and T4 to inhibit transport through human 4F2hc-LAT2. The LAT1 and LAT2 assays are of particular interest to determine possible implications and influences of 4F2hc in ligand binding and transport. In summary, the presented assays are valuable for characterization of ligands, e.g., towards 4F2hc-LAT1 specificity, and can also be applied for compound screening. Finally, our established approach and assay would also be applicable to other HATs and LATs of interest.

Real-time functional characterization of cationic amino acid transporters using a new FRET sensor

2015 ◽  
Vol 468 (4) ◽  
pp. 563-572 ◽  
Author(s):  
Liviu Vanoaica ◽  
Alok Behera ◽  
Simone M. R. Camargo ◽  
Ian C. Forster ◽  
François Verrey
Keyword(s):  
Amino Acid ◽  
Real Time ◽  
Functional Characterization ◽  
Amino Acid Transporters ◽  
Cationic Amino Acid

scholarly journals Biochemical and Functional Characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L.

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2876 ◽  
Author(s):  
Lin Tan ◽  
Mei Wang ◽  
Youfa Kang ◽  
Farrukh Azeem ◽  
Zhaoxi Zhou ◽  
...  
Keyword(s):  
Enzyme Assay ◽  
Molecular Mechanisms ◽  
Mangifera Indica ◽  
Functional Characterization ◽  
Citrate Buffer ◽  
Anthocyanidin Reductase ◽  
High Amino Acid ◽  
Optimum Ph

Mango (Mangifera indica L.) is abundant in proanthocyanidins (PAs) that are important for human health and plant response to abiotic stresses. However, the molecular mechanisms involved in PA biosynthesis still need to be elucidated. Anthocyanidin reductase (ANR) catalyzes a key step in PA biosynthesis. In this study, three ANR cDNAs (MiANR1-1,1-2,1-3) were isolated from mango, and expressed in Escherichia coli. In vitro enzyme assay showed MiANR proteins convert cyanidin to their corresponding flavan-3-ols, such as (−)-catechin and (−)-epicatechin. Despite high amino acid similarity, the recombinant ANR proteins exhibited differences in enzyme kinetics and cosubstrate preference. MiANR1-2 and MiANR1-3 have the same optimum pH of 4.0 in citrate buffer, while the optimum pH for MiANR1-1 is pH 3.0 in phosphate buffer. MiANR1-1 does not use either NADPH or NADH as co-substrate while MiANR1-2/1-3 use only NADPH as co-substrate. MiANR1-2 has the highest Km and Vmax for cyanidin, followed by MiANR1-3 and MiANR1-1. The overexpression of MiANRs in ban mutant reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate MiANRs can form the ANR pathway, leading to the formation of two types of isomeric flavan-3-ols and PAs in mango.

scholarly journals Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Travis J. Wiles ◽  
Elena S. Wall ◽  
Brandon H. Schlomann ◽  
Edouard A. Hay ◽  
Raghuveer Parthasarathy ◽  
...  
Keyword(s):  
Comparative Study ◽  
Molecular Mechanisms ◽  
Genetic Manipulation ◽  
Functional Characterization ◽  
Symbiotic Bacteria ◽  
Bacterial Isolates ◽  
Major Barrier ◽  
Allelic Exchange ◽  
Genetic Techniques

ABSTRACTCorrelating the presence of bacteria and the genes they carry with aspects of plant and animal biology is rapidly outpacing the functional characterization of naturally occurring symbioses. A major barrier to mechanistic studies is the lack of tools for the efficient genetic manipulation of wild and diverse bacterial isolates. To address the need for improved molecular tools, we used a collection of proteobacterial isolates native to the zebrafish intestinal microbiota as a testbed to construct a series of modernized vectors that expedite genetic knock-in and knockout procedures across lineages. The innovations that we introduce enhance the flexibility of conventional genetic techniques, making it easier to manipulate many different bacterial isolates with a single set of tools. We developed alternative strategies for domestication-free conjugation, designed plasmids with customizable features, and streamlined allelic exchange using visual markers of homologous recombination. We demonstrate the potential of these tools through a comparative study of bacterial behavior within the zebrafish intestine. Live imaging of fluorescently tagged isolates revealed a spectrum of distinct population structures that differ in their biogeography and dominant growth mode (i.e., planktonic versus aggregated). Most striking, we observed divergent genotype-phenotype relationships: several isolates that are predicted by genomic analysis andin vitroassays to be capable of flagellar motility do not display this trait within living hosts. Together, the tools generated in this work provide a new resource for the functional characterization of wild and diverse bacterial lineages that will help speed the research pipeline from sequencing-based correlations to mechanistic underpinnings.IMPORTANCEA great challenge in microbiota research is the immense diversity of symbiotic bacteria with the capacity to impact the lives of plants and animals. Moving beyond correlative DNA sequencing-based studies to define the cellular and molecular mechanisms by which symbiotic bacteria influence the biology of their hosts is stalling because genetic manipulation of new and uncharacterized bacterial isolates remains slow and difficult with current genetic tools. Moreover, developing tools de novo is an arduous and time-consuming task and thus represents a significant barrier to progress. To address this problem, we developed a suite of engineering vectors that streamline conventional genetic techniques by improving postconjugation counterselection, modularity, and allelic exchange. Our modernized tools and step-by-step protocols will empower researchers to investigate the inner workings of both established and newly emerging models of bacterial symbiosis.

scholarly journals Regiodivergent Biocatalytic Hydroxylation of L-Glutamine Facilitated by Characterization of Non-Heme Dioxygenases from Non-Ribosomal Peptide Biosyntheses

2021 ◽  
Author(s):  
Hans Renata ◽  
Emily Shimizu ◽  
Christian Zwick
Keyword(s):  
Amino Acid ◽  
Building Block ◽  
Solid Phase ◽  
Functional Characterization ◽  
Free Standing ◽  
Substrate Specificities ◽  
Total Turnover

We report the functional characterization of two iron- and a-ketoglutarate-dependent dioxygenases that are capable of hydroxylating free-standing glutamine at its C3 and C4 position respectively. In particular, the C4 hydroxylase, Q4Ox, catalyzes the reaction with approximately 4,300 total turnover numbers, facilitating synthesis of a solid-phase compatible building block and stereochemical elucidation at the C4 position of the hydroxylated product. This work will enable the development of novel synthetic strategies to prepare useful glutamine derivatives and stimulate further discoveries of new amino acid hydroxylases with distinct substrate specificities.<br>

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